The overall goal of this project is to advance lentiviral gene delivery systems to a point where they can serve as safe and efficient therapeutic gene delivery systems for human clinical trials. Lentiviral vectors based upon human immunodeficiency virus type I (HIV-1) and equine infectious anemia virus (EIAV) will be studied. Specifically, we are interested in improving the production and bio-safety of lentiviral vectors so that they can be used in treatments for diseases such as hemophilia B and cystic fibrosis. Thus our studies will focus on vector development and testing aspects of gene delivery to the liver and airway epithelium. It is clear that the ability of lentiviruses to transduce non-dividing cells is the main reason for development of lentivirus based gene delivery systems. Lentivirus vectors have proved efficient at transducing various tissues in vivo (brain, liver, muscle, retina, and hematopoietic stem cells). Recently, we showed that a single intraperitoneal injection of hemophilic mice with lentivirus vectors resulted in long term expression of therapeutic levels of canine factor IX. The treated mice demonstrated corrected blood clotting indices equivalent to those obtained from heterozygous litter mates. However, we believe that further improvement in vector production, vector safety, and in vivo transduction efficiencies of non-dividing cells can be made. The ability to study different lentivirus vectors in parallel will allow us to identify and to solve basic biological problems common to all lentivirus vectors. This approach permits us to investigate the effects of vector origin on the ability to efficiently transduce and express transgenes in tissues and cells from different species. To facilitate generation of cell lines for vector production, we will optimize the incorporation of Self-Inactivating (SIN) vector cassettes into stable packaging cell lines. To improve the safety of lentivirus vectors we propose to generate novel non-integrating lentiviral vectors that exhibit high levels of transgene expression in vivo. To investigate the effects of vector origin on the ability to transduce non-dividing cells, we will compare vectors derived from primate and non-primate lentiviruses on the ability to transduce non-dividing cells in model systems relevant for treatment of hemophilia B and cystic fibrosis.